Neurodegeneration and peripheral neuropathy are the major clinical consequence of human mutations affecting biosynthesis of the low abundance signaling lipid PI(3,5)P2. We have identified human and mouse mutations in the 3 major proteins of the PI(3,5)P2 biosynthetic complex: the phosphatase FIG4, the phosphokinase FAB1 (gene symbol PIKFYVE), and the scaffold protein VAC14. Neuronal expression of FIG4 is both necessary and sufficient to rescue neurodegeneration in brain and peripheral ganglia. Patients with Charcot-Marie-Tooth type 4J are compound heterozygotes for FIG4 mutations. The most common pathogenic variant in these patients is FIG4-I41T, which has an allele frequency of 0.001 in controls and destabilizes the protein, We will examine new patient cohorts by NextGen sequencing of pooled amplified exons, and carry out functional tests of rare patient variants of FIG4, VAC14 and FAB1 in cultured cells from mutant mice. As proof of principle for potential therapeutic interventions, we will examine the in vivo effects of overexpression of the transcription factor TFEB that regulates genes in the lysosome autophagy pathway. We will also evaluate treatment by up-regulation of the lysosomal cation transporter TRPML1, an effector of PI(3,5)P2 that rescues vacuolization of cultured fibroblasts from VAC14 null mice and is a potentially druggable target. We will use our floxed allele of Fig4 to distinguish the clinical contributions of motor and sensory neurons by conditional inactivation. We will investigate the functional properties of a novel mutation of FAB1 discovered in a large pedigree with Charcot-Marie-Tooth disease, as well as a novel variant of FIG4 discovered in a consanguineous pedigree with the neuronal migration disorder polymicrogyria. We will investigate the pathogenesis of intestinal obstruction in Fig4 deficient mice and its relevance to human patients. We will pinpoint the critical sites of PI(3,5)P2 dependence in the endocytosis lysosome pathway using markers of melanosome biosynthesis to dissect the pathway. The proposed experiments will integrate genetic analysis of pathogenic human mutations with functional dissection of PI(3,5)P2 activity to advance our understanding of the role of this pathway in human disease. PUBLIC HEALTH RELEVANCE: Neurons are long-lived cells that require robust protein turnover for maintenance of their network of axons and dendrites. Lysosomes and autophagy are important cellular components for protein turnover. We discovered that mutations in the gene FIG4 reduce protein turnover and neuronal survival. Patients with Charcot-Marie-Tooth Disease have mutations in FIG4. We developed mouse models of Charcot-Marie-Tooth disease that we will use to test two novel therapeutic targets. We will screen patients for defects in this pathway, and carry out functional testing of patient mutations, to test their role in this new clas of inherited neurological disorder.